Current assays for monitoring cancer generally measure the levels of tumor-associated antigens. Such assays frequently measure mucins and other glycoproteins or proteins apparently shed or secreted from the tumor into biological fluids such as serum. Tumors may also enhance the release of antigens from normal tissues into the blood stream so that levels rise during malignancy (e.g., CA 19-9 antigen). Common serological assays for tumor-associated antigens have been reviewed by Feller et al., in Immunodiagnosis of Cancer, pp 631-672 (Herberman and Mercer, eds. 1990) and in Malignant Diseases, pp. 167-294 (Baldwin, ed. 1987). Examples of such tests in general clinical use include those for carcinoembryonic antigen (CEA), CA 19-9, Ca 195, CA 125, B72.3 (TAG 72, Sialyl Tn), CA 50, CA 549, Du Pan-2 and CA 15-3 antigens. Less commonly; measured tumor-associated antigen markers include those for Le.sup.x, dimeric Le.sup.x, trimeric Le.sup.x, CS Le.sup.x, sialyl Le.sup.x -i, disialyl Le.sup.a , Le.sup.y, i, T and Tn antigens. The vast majority of tumor-associated antigens presently measured exhibit alterations in carbohydrate expression and are classified as tumor-associated carbohydrate antigens (reviewed in Hakomori, Adv. Cancer Res. 52, 257-33 (1989)).
The currently employed measurements of tumor-associated antigens are of limited clinical utility because the levels of antigens they detect generally rise over the course of the disease and are most detectable in the later stages of cancer, but are infrequently elevated in the early stages of neoplastic disease. This association of tumor-associated antigen level with tumor burden and staging may account for the relatively poor ability such tests exhibit in the prediction of cancer or cancer reoccurrence, relegating the clinical application of such assays to a monitoring role. There are exceptional cases in which tumor antigen measurements are of high diagnostic value for early stage disease (Linsley et al., Cancer Res., 46, 5444-5450 (1986)).
An alternative to measuring cancer antigen levels is to measure the immune response to tumor-associated antigens. For instance, Tal and associates (Tal et al., Br. J. Cancer, 18, 111-119 (1964)) have demonstrated that the sera from tumor patients and pregnant women contain antibodies that agglutinate tumor cells. Since lactose inhibited the agglutination, it was suggested that the antibodies were directed to lactosylceramide. Subsequently, elevated levels of antibody directed to lactosyl sphingosine were detected in a number of patients with gastrointestinal cancer (Jozwiak and Koscielak, Eur. J. Cancer Clin. Oncol., 18, 617-621 (1982)).
The specificity of human monoclonal antibodies derived from cancer patients has also been examined (reviewed in Lloyd, et. al., Cancer Res., 49, 3445-3451 (1989)). These human monoclonal antibodies were found to exhibit specificities to gangliosides (GM3, GM2, GD3, GD2), Forssman antigen, galactosyl-globoside, lacto-N-tetraose, and i antigen.
The presence of antibody to tumor-associated antigens in cancer patients has been investigated for a number of other tumor markers. These tumor markers include CEA, Le.sup.x, T and Tn antigens.
CEA is a glycoprotein containing multiple antigenic epitopes on both the oligosaccharide and peptide portions of the molecule. It is an embryonic antigen not normally present on adult tissues, but it is found in all human digestive system cancers. Thus, the presence of CEA on tissue, or the presence of circulating CEA, may be indicative of digestive system cancer. Whether antibodies to CEA are found in normals or cancer hosts has been controversial. See, Staab et al., Br. J. Cancer, 42, 26 (1980); Frenoy et al., Anticancer Research, 7, 1229 (1987). It has been reported that anti-CEA antibodies are found in patients with non-metastatic digestive system cancer and in pregnant and postpartum women, but not in normals or those with metastatic cancer. Gold, Cancer, 20, 1663 (October 1967). These results have been refuted by others. See Lo Gerfo et al., Int. J. Cancer, 9, 344 (1972); Collatz et al., Int. J. Cancer, 8, 298 (1971).
MacSween has reported the detection of antibodies to CEA, but suggests that the binding to CEA represents the cross-reactivity of antibodies to similar antigens, such as blood group antigens. MacSween, Int. J. Cancer, 15, 246 (1975). In fact, elevated levels of anti-CEA antibodies directed to Lewis.sup.ab substances on a CEA preparation have been found in women, smokers and 7-23% of patients having colonic, breast and bronchogenic carcinoma. Pompecki et al., Cancer Research, 41, 1910-1915 (1981). Pompecki et al. suggest that an increase in the levels of these antibodies is a poor prognostic sign for cancer patients and that following the levels of antibodies to blood group determinants, and perhaps other carbohydrate determinants, may be useful in monitoring disease progress in certain types of cancer and other diseases. Id.
Another group has reported circulating anti-CEA IgG antibodies in a very small number (3/500,000) of sera obtained from cancer patients. Ura et al. Cancer Letters, 25, 283 (1985). More recently, anti-CEA antibodies of weak affinity directed to the peptide portion of CEA were found in all normal and pathological sera tested using an enzyme immunoassay. Frenoy et al., Anticancer Research, 7, 1229 (1987). Finally, circulating CEA immune complexes have been described. Staab et al., Br. J. Cancer, 42, 26 (1980). Staab et al. teach that the presence of CEA immune complexes is a poor prognostic indicator for cancer patients. Id.
T and Tn are also tumor-associated antigens. While T and Tn are present on most tissues, they are normally occluded by covering structures which make them inaccessible to the immune system. T and Tn in unmasked immunoreactive form have been reported to be abundantly expressed in about 90% of carcinoma tissues. Springer, Science, 224, 1198-1206 (1984). The relative proportion and density of T and Tn on tumor surfaces have also been reported to correlate with tumor aggressiveness and invasiveness. Id. In particular, Tn is believed to be an important antigen in carcinoma metastasis. Id. All humans are reported to have antibodies against T and Tn which are thought to be elicited predominantly in response to antigens of the intestinal flora. Springer, Science, 224, 1198-1206 (1984). There have been reports that levels of anti-T are depressed, elevated or unchanged in cancer patients. See Bray et al., Clin. Exp. Immunol., 47, 176-182 (1982).
For instance, U.S. Pat. No. 4,241,044 (Kim) teaches that carcinoma patients generally have elevated levels of anti-T. However, Bray et al. teach that patients with metastatic gastrointestinal cancer have depressed serum levels of anti-T and that the levels of anti-T correlate with the level of disease in that 83% of patients with extensive disease had lower than normal levels of anti-T as compared to 45% of patients with moderate disease and none with minimal disease. Bray et al., Clin. Exp. Immunol., 47, 176-182 (1982).
It has also been reported that patients with carcinoma generally have depressed levels of anti-T as compared to normals or to patients with benign diseases, but that in rare instances patients with carcinoma have elevated levels of anti-T. Springer, Science, 224, 1198-1206 (1984); Springer et al. in Cellular Oncology: New approaches in Biology, Diagnosis and Treatment, pages 99-130 (Moloy and Nicolson eds., 1982).
Finally, Thatcher et al. teach that anti-T titers are subnormal in patients with disseminated melanoma before therapy, and that patients who responded to therapy had significantly higher titers than did nonresponders in sera taken before therapy, at regression or progression of disease, and during the last pulse of treatment. Thatcher et al., Cancer, 46, 1378 (1980). Higher pretreatment titers were also associated with a significantly longer survival time. Id.
N3 is reported to be a mixture of difucosyl lacto-N-hexaoses and difucosyl lacto-N-neohexaoses which is isolated from breast milk. Kobata and Ginsburg, Arch. Biochem. Biophys., 150, 273-281 (1972). Lacto-N-hexaose has the formula: Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.6(Gal.beta.1.fwdarw.3GlcNAc.beta.1 .fwdarw.3)Gal.beta.1 .fwdarw.4Glc. Id. Lacto-N-neohexaose had the formula: Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.6(Gal.beta.1.fwdarw.4GlcNAc.beta.1 .fwdarw.3)Gal.beta.1.fwdarw.4Glc. Id. Kobata and Ginsburg speculate that the two fucose residues may be attached .alpha.1.fwdarw.2 to galactose, .alpha.1.fwdarw.3 to N-acetylglucosamine, .alpha.1.fwdarw.4 to N-acetylglucosamine, or .alpha.1.fwdarw.3 to glucose. Id. The exact composition of N3 will vary depending on the Lewis blood type of the donor. Id.
N3 isolated from the breast milk of individuals of Lewis blood type Le(a+b-) contains an octasaccharide having the following formula I: ##STR1## Dua et al., J. Chrom., 17, 259-269 (1985). This octasaccharide is the predominant isomer, comprising at least 90% of the N3 isolated from Lewis (a+b-) individuals. Id. Dua et al. were unable to demonstrate the presence of difucosyl lacto-N-neohexaoses in their N3 preparation. Id. Based on the limits of their methodology they, therefore, concluded that N3 isolated from Lewis (a+b-) individuals contained at most 10% of such difucosyl lacto-N-neohexaoses. Id. Note that N3 is referred to as M3 in the Dua et al. article.
The immunodeterminant structure of the Le.sup.a blood group antigen is the trisaccharide Gal.beta.1.fwdarw.3(Fuc.alpha.1.fwdarw.4)GlcNAc, which is carried by a type 1 blood group chain. The immunodeterminant structure of Le.sup.x is the trisaccharide Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc which is carried by a type 2 blood group chain. Le.sup.x is also sometimes designated 3-fucosyl-N-acetyl-lactosamine, X hapten, Cluster of Differentiation (CD) 15 leukocyte antigen in humans, and stage-specific embryonic antigen 1 (SSEA-1) in mice. As can be seen from an examination of the above formula I, N3 isolated from Lewis (a+b-) individuals contains Le.sup.a and Le.sup.x determinants. Accordingly, as would be expected, N3 isolated from Lewis (a+b-) individuals displays Le.sup.a and Le.sup.x hapten activity in hemagglutination assays. Id.; Rohr et al., Fed. Proc., 42, 2102 (1983); Rohr et al., Fed. Proc., 42, 431 (1983); Rohr et al., Soc. Complex Carbohydrates, October 1983.
In epithelial-derived cancers of the colon and pancreas, the Le.sup.a blood group antigen is not considered a tumor-associated antigen because of its prominent expression in both normal and cancer tissues. Itzkowitz, et al., Cancer Res., 48, 3834-3842 (1988); Yuan, et al., Cancer Res., 45, 4499-4511 (1985); Ernst, et al., Lab. Invest., 50, 394-400 (1984). Le.sup.x is present on a variety of normal cells such as kidney tubules, gastrointestinal epithelia and granulocytes, and it accumulates in a variety of human cancers including those of leukocyte, stomach, liver, lung, and colon origin. Gooi, et al., Eur. J. Immunol., 13, 306-312 (1983); Hakomori, Adv. Cancer Res., 52, 257-331 (1989); Hakomori, J. Nat. Cancer Inst., 71, 231-250 (1983).
For instance, Le.sup.x antigen accumulates in gastrointestinal cancers in both membrane-bound forms (glycolipids and glycoproteins) and secreted forms (glycoproteins and mucins). The membrane-bound forms are generally detected by immunocytochemical means, while the secreted forms are assayed using standard immunoassay methods.
The expression of Le.sup.x antigen on tissue occurs at a higher frequency in early gastric cancer (93.3%) than in advanced stage cancer (66.7%), and Le.sup.x is present in 81% of cases of histologically classified undifferentiated gastric cancer and 66.7% of differentiated gastric cancer. Dohi et al., Gastroenterol. Jpm, 24, 239-245 (1989). Increased levels of the expression of Le.sup.x antigen on tissues have also been observed in pancreatic cancer and colorectal adenocarcinomas. Schuessler et al., Int. J. Cancer, 47, 180-187 (1991); Shi et al., Cancer Res., 44, 1142-1147 (1984); Itzkowitz et al., Cancer Res., 46, 2627-2632 (1986).
Immunoassays measuring levels of Le.sup.x in the sera of cancer patients have given mixed results. Kannagi et al., Cancer Res., 46, 2619-2626 (1986) report that levels of Le.sup.x and polyLe.sup.x, while occasionally high, were elevated in only about 10% of the cancer sera tested. These authors also reported that serum levels of sialyl Le.sup.x -i were significantly high in patients with cancers originating from organs from which adenocarcinomas often develop, and that the serum levels of Le.sup.y were frequently high in patients with hepatoma. They concluded that all four antigens (Le.sup.x, polyLe.sup.x, sialyl Le.sup.x -i and Le.sup.y) were tumor-associated antigens.
Herlyn et al., J. Immunol. Methods, 75, 15-21 (1984) reports that 53% of colorectal cancer patients older than 65 years had elevated levels of antibodies to lacto-N-fucopentaose (LNF) III compared to none of age-matched, apparently healthy donors or patients with benign gastrointestinal tract lesions and 18% of patients with inflammatory gastrointestinal tract diseases. In younger patients, the differences were less marked, and LNF III is found at relatively high levels in normals under 25 years of age. LNF III is a carbohydrate containing a Le.sup.x determinant.
A good correlation between Le.sup.x levels and the stage of colon cancer has been reported when two high affinity monoclonal antibodies directed to mono-Le.sup.x and dimeric-Le.sup.x structures were used for the assay of Le.sup.x levels in colorectal adenocarcinoma sera. Singhal et al., Cancer Res., 50, 1375-1380 (1990). Singhal et al. report that the preoperative levels of serum Le.sup.x increased in association with the progression of colorectal cancer (Dukes stages A to D) after surgery. In particular, they found that the percentage of serum Le.sup.x above the upper normal control level of 10 ng/ml were as follows:
______________________________________ Dukes A 20% Dukes B, with recurrent cancer 41% Dukes B, without recurrent cancer 46% Dukes C, with recurrent cancer 68% Dukes C, without recurrent cancer 67% Dukes D 74%. ______________________________________
A longitudinal study of Dukes B and C patients having Le.sup.x levels of greater than 20 ng/ml was performed by these researchers. In Dukes C patients exhibiting cancer recurrence, postoperative Le.sup.x levels rose significantly twenty-four months after surgery, while Dukes C patients without cancer recurrence showed a decrease in Le.sup.x antigen levels over the same period. In Dukes B patients, postoperative levels of Le.sup.x did not rise in patients with or without recurrence of cancer over the same twenty-four month period. The Le.sup.x antigen detected by these researchers was expressed on a large molecular weight glycoprotein (M.sub.r about 200,000). This glycoprotein is apparently different from the glycoproteins bearing the CEA or sialyl-Le.sup.a (19-9) antigens. The Le.sup.x glycoprotein was shown to be present in 85% of the sera of adenocarcinoma patients and in 33% of normal sera. The authors suggest that serum Le.sup.x antigen levels could be of diagnostic and prognostic value in adenocarcinoma.
The existence of human antibodies to Le.sup.x has been demonstrated by the isolation of circulating immune complexes (CIC) from the sera of adenocarcinoma patients which, upon dissociation, were shown to contain antibodies reactive with purified glycolipids bearing Le.sup.x antigen and an antigen component expressing Le.sup.x antigen(s). Singhal et al., Cancer Res., 47, 5566-5571 (1987). These investigators found that significantly higher levels of these CICs could be detected in patients suffering from breast, lung and colon adenocarcinoma as compared to controls (normals and melanoma patients) and that these CICs were detected in much higher percentages of adenocarcinoma patients than was the sialyl-Le.sup.x antigen.
Thus, while N3 itself has not been demonstrated to be a tumor-associated antigen, the Le.sup.x determinant, which is part of the N3 oligosaccharide, is a tumor-associated structure preferentially expressed on tumors. See, e.g., Davis et al., Fed. Proc., 43, 1751 (1984); Anderson et al., in The Molecular Immunology of Complex Carbohydrates, pp. 601-656 (Wu, ed. 1988); Hakomori and Kannagi, "Carbohydrate Antigens in Higher Animals," in Handbook of Experimental Immunology, Volume 1, pp. 9.1-9.39, (Weir, ed. 1986); Pompecki et al., Cancer Research, 41, 1910-1915 (1981). Also, significantly higher levels of CICs containing antibodies to Le.sup.x could be detected in adenocarcinoma patients as compared to controls. However, Applicants are not aware of any reports of the presence or changed levels of uncomplexed antibodies to Le.sup.x in cancer patients.